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Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.

ABSTRACT: The NF-κB essential modulator NEMO is the core regulatory component of the inhibitor of κB kinase complex, which is a critical checkpoint in canonical NF-κB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1β, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-1β. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional relevance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is defective in mediating IL-1β-induced NF-κB activation.

SUBMITTER: Goel S

PROVIDER: S-EPMC9889916 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.

Goel Simran S Oliva Rosario R Jeganathan Sadasivam S Bader Verian V Krause Laura J LJ Kriegler Simon S Stender Isabelle D ID Christine Chadwick W CW Nakamura Ken K Hoffmann Jan-Erik JE Winter Roland R Tatzelt Jörg J Winklhofer Konstanze F KF

Life science alliance 20230131 4

The NF-κB essential modulator NEMO is the core regulatory component of the inhibitor of κB kinase complex, which is a critical checkpoint in canonical NF-κB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1β, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and ...[more]

PMID: 36720498

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Project description:Molluscum contagiosum virus (MCV) is a human-adapted poxvirus that causes a common and persistent yet mild infection characterized by distinct, contagious, papular skin lesions. These lesions are notable for having little or no inflammation associated with them and can persist for long periods without an effective clearance response from the host. Like all poxviruses, MCV encodes potent immunosuppressive proteins that perturb innate immune pathways involved in virus sensing, the interferon response, and inflammation, which collectively orchestrate antiviral immunity and clearance, with several of these pathways converging at common signaling nodes. One such node is the regulator of canonical nuclear factor kappa B (NF-κB) activation, NF-κB essential modulator (NEMO). Here, we report that the MCV protein MC008 specifically inhibits NF-κB through its interaction with NEMO, disrupting its early ubiquitin-mediated activation and subsequent downstream signaling. MC008 is the third NEMO-targeting inhibitor to be described in MCV to date, with each inhibiting NEMO activation in distinct ways, highlighting strong selective pressure to evolve multiple ways of disabling this key signaling protein. IMPORTANCE Inflammation lies at the heart of most human diseases. Understanding the pathways that drive this response is the key to new anti-inflammatory therapies. Viruses evolve to target inflammation; thus, understanding how they do this reveals how inflammation is controlled and, potentially, how to disable it when it drives disease. Molluscum contagiosum virus (MCV) has specifically evolved to infect humans and displays an unprecedented ability to suppress inflammation in our tissue. We have identified a novel inhibitor of human innate signaling from MCV, MC008, which targets NEMO, a core regulator of proinflammatory signaling. Furthermore, MC008 appears to inhibit early ubiquitination, thus interrupting later events in NEMO activation, thereby validating current models of IκB kinase (IKK) complex regulation.

Project description:Viruses have adopted diverse strategies to suppress antiviral responses. Hepatitis B virus (HBV), a virus that is prevalent worldwide, manipulates the host's innate immune system to evade scavenging. It is reported that the hepatitis B e antigen (HBeAg) can interfere with NF-κB activity, which then leads to high viral loads, while HBV with the G1896A mutation remains infectious without the production of HBeAg but can induce more severe proinflammatory response and liver damage. The aim of current work was to study the molecular mechanism by which HBeAg suppresses interleukin-1β (IL-1β)-stimulated NF-κB activity, which leads to the suppression of the innate immune responses to HBV infection. Our study revealed that HBeAg could interact with NEMO, a regulatory subunit associated with IκB kinase, which regulates the activation of NF-κB. HBeAg suppressed the IL-1β-induced tumor necrosis factor (TNF)-associated factor 6 (TRAF6)-dependent K63-linked ubiquitination of NEMO, thereby downregulating NF-κB activity and promoting virus replication. We further demonstrated the inhibitory effect of HBeAg on the NF-κB signaling pathway using primary human hepatocytes, HBV-infected HepG2-NTCP cells, and clinical liver samples. Our study reveals a molecular mechanism whereby HBeAg suppresses IL-1β-induced NF-κB activation by decreasing the TRAF6-dependent K63-linked ubiquitination of NEMO, which may thereby enhance HBV replication and promote a persistent infection.IMPORTANCE The role of HBeAg in inflammatory responses during the infection of hepatitis B virus (HBV) is not fully understood, and several previous reports with regard to the NF-κB pathway are controversial. In this study, we showed that HBeAg could suppress both Toll-like receptor 2 (TLR2)- and IL-1β-induced activation of NF-κB in cells and clinical samples, and we further revealed novel molecular mechanisms. We found that HBeAg can associate with NEMO, the regulatory subunit for IκB kinase (IKK) that controls the NF-κB signaling pathway, and thereby inhibits TRAF6-mediated K63-linked ubiquitination of NEMO, resulting in downregulation of NF-κB activity and promotion of virus replication. In contrast, the HBeAg-negative HBV mutant can induce higher levels of NF-κB activity. These results are important for understanding the HBV-induced pathogenesis of chronic hepatitis and indicate that different clinical measures should be considered to treat HBeAg-positive and HBeAg-negative infections. Our findings represent a conceptual advance in HBV-related suppression of NF-κB signaling.

Project description:BackgroundUTP-glucose-1-phosphoryl transferase (UGPase) catalyzes the synthesis of UDP-glucose, which is essential for generating the glycogen needed for the synthesis of bacterial lipopolysaccharide (LPS) and capsular polysaccharide, which play important roles in bacterial virulence. However, the molecular function of UGPase in Brucella is still unknown.ResultsIn this study, the ubiquitination modification of host immune-related protein in cells infected with UGPase-deleted or wild-type Brucella was analyzed using ubiquitination proteomics technology. The ubiquitination modification level and type of NF-κB Essential Modulator (NEMO or Ikbkg), a molecule necessary for NF-κB signal activation, was evaluated using Coimmunoprecipitation, Western blot, and dual-Luciferase Assay. We found 80 ubiquitin proteins were upregulated and 203 ubiquitin proteins were downregulated in cells infected with B. melitensis 16 M compared with those of B. melitensis UGPase-deleted strain (16 M-UGPase-). Moreover, the ubiquitin-modified proteins were mostly enriched in the categories of regulation of kinase/NF-κB signaling and response to a bacterium, suggesting Brucella UGPase inhibits ubiquitin modification of related proteins in the host NF-κB signaling pathway. Further analysis showed that the ubiquitination levels of NEMO K63 (K63-Ub) and Met1 (Met1-Ub) were significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M-infected cells, further confirming that the ubiquitination levels of NF-κB signaling-related proteins were regulated by the bacterial UGPase. Besides, the expression level of IκBα was decreased, but the level of p-P65 was significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M- and mock-infected cells, demonstrating that B. melitensis UGPase can significantly inhibit the degradation of IκBα and the phosphorylation of p65, and thus suppressing the NF-κB pathway.ConclusionsThe results of this study showed that Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO, which will provide a new scientific basis for the study of immune mechanisms induced by Brucella.

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Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.

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Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.

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